BMP9-induced osteoblastic differentiation requires functional Notch signaling in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into multiple lineages including osteoblastic lineage. Osteogenic differentiation of MSCs is a cascade that recapitulates most, if not all, of the molecular events occurring during embryonic skeletal development, which is regulated by numerous signaling pathways including bone morphogenetic proteins (BMPs). Through a comprehensive analysis of the osteogenic activity, we previously demonstrated that BMP9 is the most potent BMP for inducing bone formation from MSCs both in vitro and in vivo. However, as one of the least studied BMPs, the essential mediators of BMP9-induced osteogenic signaling remain elusive. Here we show that BMP9-induced osteogenic signaling in MSCs requires intact Notch signaling. While the expression of Notch receptors and ligands are readily detectable in MSCs, Notch inhibitor and dominant-negative Notch1 effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic bone formation in vivo. Genetic disruption of Notch pathway severely impairs BMP9-induced osteogenic differentiation and ectopic bone formation from MSCs. Furthermore, while BMP9-induced expression of early-responsive genes is not affected by defective Notch signaling, BMP9 upregulates the expression of Notch receptors and ligands at the intermediate stage of osteogenic differentiation. Taken together, these results demonstrate that Notch signaling may play an essential role in coordinating BMP9-induced osteogenic differentiation of MSCs.

Reversibly immortalized human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are responsive to BMP9-induced osteogenic and adipogenic differentiation.

Human mesenchymal stem cells (MSCs) are a heterogeneous subset of nonhematopoietic multipotent stromal stem cells and can differentiate into mesodermal lineage, such as adipocytes, osteocytes, and chondrocytes, as well as ectodermal and endodermal lineages. Human umbilical cord (UC) is one of the most promising sources of MSCs. However, the molecular and cellular characteristics of UC-derived MSCs (UC-MSCs) require extensive investigations, which are hampered by the limited lifespan and the diminished potency over passages. Here, we used the piggyBac transposon-based simian virus 40 T antigen (SV40T) immortalization system and effectively immortalized UC-MSCs, yielding the iUC-MSCs. A vast majority of the immortalized lines are positive for MSC markers but not for hematopoietic markers. The immortalization phenotype of the iUC-MSCs can be effectively reversed by flippase recombinase-induced the removal of SV40T antigen. While possessing long-term proliferation capability, the iUC-MSCs are not tumorigenic in vivo. Upon bone morphogenetic protein 9 (BMP9) stimulation, the iUC-MSC cells effectively differentiate into osteogenic, chondrogenic, and adipogenic lineages both in vitro and in vivo, which is indistinguishable from that of primary UC-MSCs, indicating that the immortalized UC-MSCs possess the characteristics similar to that of their primary counterparts and retain trilineage differentiation potential upon BMP9 stimulation. Therefore, the engineered iUC-MSCs should be a valuable alternative cell source for studying UC-MSC biology and their potential utilities in immunotherapies and regenerative medicine.

Niclosamide Exhibits Potent Anticancer Activity and Synergizes with Sorafenib in Human Renal Cell Cancer Cells.

BACKGROUND/AIMS: As the most lethal urological cancers, renal cell carcinoma (RCC) comprises a heterogeneous group of cancer with diverse genetic and molecular alterations. There is an unmet clinical need to develop efficacious therapeutics for advanced, metastatic and/or relapsed RCC. Here, we investigate whether anthelmintic drug Niclosamide exhibits anticancer activity and synergizes with targeted therapy Sorafenib in suppressing RCC cell proliferation. METHODS: Cell proliferation and migration were assessed by Crystal violet staining, WST-1 assay, cell wounding and cell cycle analysis. Gene expression was assessed by qPCR. In vivo anticancer activity was assessed in xenograft tumor model. RESULTS: We find that Niclosamide effectively inhibits cell proliferation, cell migration and cell cycle progression, and induces apoptosis in human renal cancer cells. Mechanistically, Niclosamide inhibits the expression of C-MYC and E2F1 while inducing the expression of PTEN in RCC cells. Niclosamide is further shown to synergize with Sorafenib in suppressing RCC cell proliferation and survival. In the xenograft tumor model, Niclosamide is shown to effectively inhibit tumor growth and suppress RCC cell proliferation. CONCLUSIONS: Niclosamide may be repurposed as a potent anticancer agent, which can potentiate the anticancer activity of the other agents targeting different signaling pathways in the treatment of human RCC.

Corrigendum to "The development of a sensitive fluorescent protein-based transcript reporter for high throughput screening of negative modulators of lncRNAs" [Genes & Diseases 5 (2018) 62-74].

[This corrects the article DOI: 10.1016/j.gendis.2018.02.001.].

Corrigendum to "Characterization of the essential role of bone morphogenetic protein 9 (BMP9) in osteogenic differentiation of mesenchymal stem cells (MSCs) through RNA interference" [Genes & Diseases 5(2018):172-184].

[This corrects the article DOI: 10.1016/j.gendis.2018.04.006.].

A pH-Triggered, Self-Assembled, and Bioprintable Hybrid Hydrogel Scaffold for Mesenchymal Stem Cell Based Bone Tissue Engineering.

Effective bone tissue engineering can restore bone and skeletal functions that are impaired by traumas and/or certain medical conditions. Bone is a complex tissue and functions through orchestrated interactions between cells, biomechanical forces, and biofactors. To identify ideal scaffold materials for effective mesenchymal stem cell (MSC)-based bone tissue regeneration, here we develop and characterize a composite nanoparticle hydrogel by combining carboxymethyl chitosan (CMCh) and amorphous calcium phosphate (ACP) (designated as CMCh-ACP hydrogel). We demonstrate that the CMCh-ACP hydrogel is readily prepared by incorporating glucono δ-lactone (GDL) into an aqueous dispersion or rehydrating the acidic freeze-dried nanoparticles in a pH-triggered controlled-assembly fashion. The CMCh-ACP hydrogel exhibits excellent biocompatibility and effectively supports MSC proliferation and cell adhesion. Moreover, while augmenting BMP9-induced osteogenic differentiation, the CMCh-ACP hydrogel itself is osteoinductive and induces the expression of osteoblastic regulators and bone markers in MSCs in vitro. The CMCh-ACP scaffold markedly enhances the efficiency and maturity of BMP9-induced bone formation in vivo, while suppressing bone resorption occurred in long-term ectopic osteogenesis. Thus, these results suggest that the pH-responsive self-assembled CMCh-ACP injectable and bioprintable hydrogel may be further exploited as a novel scaffold for osteoprogenitor-cell-based bone tissue regeneration.

A Simplified System to Express Circularized Inhibitors of miRNA for Stable and Potent Suppression of miRNA Functions.

MicroRNAs (miRNAs) are an evolutionarily conserved class of small regulatory noncoding RNAs, binding to complementary target mRNAs and resulting in mRNA translational inhibition or degradation, and they play an important role in regulating many aspects of physiologic and pathologic processes in mammalian cells. Thus, efficient manipulations of miRNA functions may be exploited as promising therapeutics for human diseases. Two commonly used strategies to inhibit miRNA functions include direct transfection of chemically synthesized miRNA inhibitors and delivery of a gene vector that instructs intracellular transcription of miRNA inhibitors. While most miRNA inhibitors are based on antisense molecules to bind and sequester miRNAs from their natural targets, it is challenging to achieve effective and stable miRNA inhibition. Here we develop a user-friendly system to express circular inhibitors of miRNA (CimiRs) by exploiting the noncanonical head-to-tail backsplicing mechanism for generating endogenous circular RNA sponges. In our proof-of-principle experiments, we demonstrate that the circular forms of the hsa-miR223-binding site of human ß-arrestin1 (ARRB1) 3' UTR sponge RNA (BUTR), the bulged anti-miR223 (cirBulg223) and bulged anti-miR21 (cirBulg21), exhibit more potent suppression of miRNA functions than their linear counterparts. Therefore, the engineered CimiR expression system should be a valuable tool to target miRNAs for basic and translational research.

The development of a sensitive fluorescent protein-based transcript reporter for high throughput screening of negative modulators of lncRNAs.

While the human genome is pervasively transcribed, <2% of the human genome is transcribed into protein-coding mRNAs, leaving most of the transcripts as noncoding RNAs, such as microRNAs and long-noncoding RNAs (lncRNAs), which are critical components of epigenetic regulation. lncRNAs are emerging as critical regulators of gene expression and genomic stability. However, it remains largely unknown about how lncRNAs are regulated. Here, we develop a highly sensitive and dynamic reporter that allows us to identify and/or monitor negative modulators of lncRNA transcript levels in a high throughput fashion. Specifically, we engineer a fluorescent fusion protein by fusing three copies of the PEST destruction domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of the codon-optimized bilirubin-inducible fluorescent protein, designated as dBiFP, and show that the dBiFP protein is highly destabilized, compared with the commonly-used eGFP protein. We further demonstrate that the dBiFP signal is effectively down-regulated when the dBiFP and mouse lncRNA H19 chimeric transcript is silenced by mouse H19-specific siRNAs. Therefore, our results strongly suggest that the dBiFP fusion protein may serve as a sensitive and dynamic transcript reporter to monitor the inhibition of lncRNAs by microRNAs, synthetic regulatory RNA molecules, RNA binding proteins, and/or small molecule inhibitors so that novel and efficacious inhibitors targeting the epigenetic circuit can be discovered to treat human diseases such as cancer and other chronic disorders.

Characterization of the essential role of bone morphogenetic protein 9 (BMP9) in osteogenic differentiation of mesenchymal stem cells (MSCs) through RNA interference.

Mesenchymal stem cells (MSCs) are multipotent stem cells and capable of differentiating into multiple cell types including osteoblastic, chondrogenic and adipogenic lineages. We previously identified BMP9 as one of the most potent BMPs that induce osteoblastic differentiation of MSCs although exact molecular mechanism through which BMP9 regulates osteogenic differentiation remains to be fully understood. Here, we seek to develop a recombinant adenovirus system to optimally silence mouse BMP9 and then characterize the important role of BMP9 in osteogenic differentiation of MSCs. Using two different siRNA bioinformatic prediction programs, we design five siRNAs targeting mouse BMP9 (or simB9), which are expressed under the control of the converging H1 and U6 promoters in recombinant adenovirus vectors. We demonstrate that two of the five siRNAs, simB9-4 and simB9-7, exhibit the highest efficiency on silencing exogenous mouse BMP9 in MSCs. Furthermore, simB9-4 and simB9-7 act synergistically in inhibiting BMP9-induced expression of osteogenic markers, matrix mineralization and ectopic bone formation from MSCs. Thus, our findings demonstrate the important role of BMP9 in osteogenic differentiation of MSCs. The characterized simB9 siRNAs may be used as an important tool to investigate the molecular mechanism behind BMP9 osteogenic signaling. Our results also indicate that recombinant adenovirus-mediated expression of siRNAs is efficient and sustained, and thus may be used as an effective delivery vehicle of siRNA therapeutics.

Thermoresponsive Citrate-Based Graphene Oxide Scaffold Enhances Bone Regeneration from BMP9-Stimulated Adipose-Derived Mesenchymal Stem Cells.

Effective bone tissue engineering is important to overcome the unmet clinical challenges as more than 1.6 million bone grafts are done annually in the United States. Successful bone tissue engineering needs minimally three critical constituents: osteoprogenitor cells, osteogenic factors, and osteoinductive/osteoconductive scaffolds. Osteogenic progenitors are derived from multipotent mesenchymal stem cells (MSCs), which can be prepared from numerous tissue sources, including adipose tissue. We previously showed that BMP9 is the most osteogenic BMP and induces robust bone formation of immortalized mouse adipose-derived MSCs entrapped in a citrate-based thermoresponsive hydrogel referred to as PPCNg. As graphene and its derivatives emerge as promising biomaterials, here we develop a novel thermosensitive and injectable hybrid material by combining graphene oxide (GO) with PPCNg (designated as GO-P) and characterize its ability to promote bone formation. We demonstrate that the thermoresponsive behavior of the hybrid material is maintained while effectively supporting MSC survival and proliferation. Furthermore, GO-P induces early bone-forming marker alkaline phosphatase (ALP) and potentiates BMP9-induced expression of osteogenic regulators and bone markers as well as angiogenic factor VEGF in MSCs. In vivo studies show BMP9-transduced MSCs entrapped in the GO-P scaffold form well-mineralized and highly vascularized trabecular bone. Thus, these results indicate that GO-P hybrid material may function as a new biocompatible, injectable scaffold with osteoinductive and osteoconductive activities for bone regeneration.